Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries

BACKGROUND: Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. METHODS: Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. RESULTS: ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. CONCLUSIONS: ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.


NOVELTY AND RELEVANCE What Is New?
A naturally occurring NO synthase inhibitor, asymmetric dimethylarginine, predisposes to vasospasm by switching small mesenteric and coronary resis tance arteries into an electrically excitable state, while vasodilator capacity is preserved by endotheliumdependent hyperpolarization.NO-dependent endothelial vasodilation was blocked by asymmetric dimethylarginine in coronary arteries but only partially inhibited in mesenteric resistance arteries.Persistent NO-vasodilation correlated with greater dimethylarginine dimethylaminohydrolase-expression in rat-isolated small mesenteric arteries endothelium and sensitivity to dimethylarginine dimethylaminohydrolase inhibition.

What Is Relevant?
NO is a potent vasodilator released by the endothelium but must also be considered in terms of its chronic ability directly to suppress arterial vasoreactivity.
Enhanced small artery vasoreactivity due in part to increased electrical excitability may be a significant feature of microvascular dysfunction.

Clinical/Pathophysiological Implications?
Coronary microvascular dysfunction is a major contributing factor in ischemic heart disease.The underlying mechanisms are complex and poorly understood.
Recognizing loss of NO as a key feature of coronary microvascular dysfunction that may arise from endogenous accumulation of endothelial asymmetric dimethylarginine suggests chronic intervention to protect NO signaling should be given serious consideration.
Possibilities for pharmacological intervention include the use of selective T-type voltage-gated calcium channel blockers to reduce vascular smooth muscle electrical excitability, outcompeting endothelial accumulation of asymmetric dimethylarginine with L-arginine or sustaining cyclic guanosine monophosphate levels with phospho diesterase inhibitors.The current study had 2 aims.First, to investigate whether, like L-NAME, the endogenous eNOS-inhibitor ADMA enables depolarizing spikes and vasospasm in both nonmyogenic rat-isolated small mesenteric arteries (RMAs) and myogenically active rat-isolated intraseptal coronary arteries (RCAs).Functional clarification is important, as nitro-arginine inhibitors such as L-NAME are reported to be more potent than methylated arginine derivatives. 15,16Second, to investigate whether endogenous metabolism by DDAH (dimethylarginine dimethylaminohydrolases) influences the functional impact of ADMA on small artery vasoreactivity.In the aorta, inhibition of DDAH raised the level of endogenous ADMA and enhanced vasoconstriction to phenylephrine. 17Our data show ADMA, like L-NAME, does enable depolarizing spikes, enhancing vasoreactivity.They also suggest the functional influence of DDAH1 varies between small arteries, as ADMA failed to block NO-mediated vasorelaxation in mesenteric resistance arteries unlike small coronary arteries.Overall, these data are consistent with the idea that the microvascular dysfunction underlying ischemic heart disease could in part reflect enhanced vasoreactivity following endogenous accumulation of ADMA.They also indicate that supplementation with L-arginine can reduce the impact of NO loss.

METHODS
Male Wistar rat small mesenteric and intraseptal coronary arteries were mounted in a Mulvany-Halpern myograph to record isometric tension with simultaneous measurement of membrane potential or VSM intracellular Ca 2+ events or in a pressure myograph. 18,19Arteries were then fixed in situ for immunohistochemistry. Data were analyzed using Microsoft Excel 2011 (Microsoft Corporation) and GraphPad Prism Software (v10.0,GraphPad Software). 20,21More detail is found in the Supplement Material, Expanded Materials and Methods.Data that support the findings of this study are available from the corresponding author upon reasonable request.

NO-Mediated Vasorelaxation to ACh Persists With ADMA in RMA
In PE-stimulated arteries with either 300 µmol/L ADMA or 100 µmol/L L-NAME present to block NO synthase, ACh evoked concentration-dependent smooth muscle hyperpolarization and vasorelaxation (an endothelium-dependent hyperpolarization (EDH) response; Figure 2A, 2B, 2E, and 2F).Endotheliumdependent mesenteric artery vasorelaxation is mediated by the parallel action of both NO and EDH, the latter generated by dual activation of EC SK Ca and IK Ca channels.Subsequent block of EDH with 1 µmol/L NS6180 (blocks EC IK Ca channels, K Ca 3.2 22 ) combined with 0.1 µmol/L apamin (blocks endothelial SK Ca channels, K Ca 2.1) abolished EDH hyperpolarization and vasorelaxation in the presence of L-NAME.However, vasorelaxation persisted with ADMA and NS6180/ apamin present, even though hyperpolarization was abolished (Figure 2C through 2F).Subsequent addition of the K ATP channel activator, 5 µmol/L levcromakalim stimulated hyperpolarization (to −72±1.0mV) and complete relaxation (to 1.2±0.2mN/mm −1 ), n=5.

Downloaded from http://ahajournals.org by on April 19, 2024
A similar profile was obtained in pressurized RMA with ADMA.Circa 50% ACh-vasodilation persisted during block of EDH (with NS6180 and apamin) and eNOS, the latter with 300 µmol/L ADMA.Loss of vasodilation was augmented in the presence of L-257.As with wire myography, ACh vasodilation could be protected on incubation with 1 mmol/L L-arginine (Figure 3E), even when DDAH1 was inhibited with L-257 (Figure 3F).

ORIGINAL ARTICLE
with the appearance of rapid depolarizing spikes (mean amplitude, 17.0±2.0mV, 1.0±0.1 Hz, maximum, 25.4±3.3mV, n=12).L-arginine (1 mmol/L) reduced both spike amplitude and associated vasoconstriction with ADMA to control levels (P>0.05; Figure 5B).Waveform analysis of membrane potential changes illustrates both variability in spike frequency, the increased power with ADMA and L-NAME, and reversal with L-arginine, summarized in Figure 5D.Tension analysis reflected the sustained vasoconstriction at each level.6F).This concentration of L-arginine reversed vasoconstriction induced by 300 µmol/L ADMA but not 100 µmol/L L-NAME (Figure S4).Lower concentrations (10 and 30 µmol/L) of ADMA significantly increased myogenic tone and 30 µmol/L ADMA inhibited, but did not block, ACh vasorelaxation (Figure S1C and S1D).
In the presence 300 µmol/L ADMA, block of EDH with NS6180 and apamin abolished ACh vasorelaxation in RCA (Figure 6E and 6F).A similar profile was obtained using 100 µmol/L L-NAME, which was augmented by EDH block (Figure 6D and 6F; Figure S5).Levcromakalim 1 µmol/L hyperpolarized and completely relaxed these arteries (with ADMA

DISCUSSION
We show the endogenous NO synthase inhibitor, ADMA, predisposes small resistance arteries to vasospasm by inducing a hyperexcitable state due to depolarizing action potential-like spikes in the VSM, rather than loss of NOdependent vasorelaxation.The latter is sustained by EDH, although reduced in the RCA where NO contributed to EDH.Once EDH was blocked the sensitivity to ADMA varied between RMA and RCA.NO-vasorelaxation was abolished in RCA, but only partially inhibited in RMA where persistent NO-vasorelaxation appeared to reflect ADMA metabolism by DDAH1.Block of endothelial NO synthase with synthetic arginine derivatives such as L-NAME markedly increases vascular reactivity.We recently discovered increased vasoreactivity is due to increased VSM electrical excitability, rather than reduced endothelial vasodilation.Raised electrical excitability enabled previously quiescent VSM to generate Ca 2+ -based depolarizing spikes with vasospasm.The spikes were reminiscent of action potentials, apart from a variable amplitude, and due to recruitment of latent T-type-VGCCs. 14We now show the endogenous methylarginine, ADMA can induce a similar change in small arteries.Previous data suggest endogenous methylarginines, N G -monomethyl L-arginine and ADMA, are not always as potent as widely used synthetic NO inhibitors, so this is an important observation. 15,16,24The interaction between ADMA and NO synthase is reversible, and our data are consistent with this, as 1 mmol/L L-arginine prevented or reversed increased VSM electrical activity, vasoreactivity, and the loss of endotheliumdependent ACh vasorelaxation with 300 µmol/L ADMA.Interestingly, RCAs were far more sensitive to ADMA than RMA, consistent with lower EC-DDAH1 expression.Importantly, increased myogenic tone with 10 µmol/L ADMA was reversed with 30 µmol/L L-arginine (Figure S1C), consistent with ≈3:1 ratio for reversing ADMA.
Small artery hyperexcitability due to VSM electrical activity may represent an important component of microvascular dysfunction.Increased RhoA/Rho kinase signaling has been linked to vasospasm in the coronary microvasculature, 25 so electrical activity causing calcium influx would interact synergistically with VSMsensitization.][28] Loss of endothelium-dependent vasorelaxation is thought central to the increased vasoreactivity with declining NO bioavailability.In small resistance arteries, endothelium-dependent vasorelaxation reflects the parallel influence of hyperpolarization (EDH) and NO release.The former is due to hyperpolarizing current generated by small conductance calcium-activated potassium channel (SK Ca ) and intermediate conductance calciumactivated potassium channels (IK Ca ) in channels in the endothelium, and block of both channel types is usually necessary to block EDH. 29In contrast to most small arteries, EDH in RCA was abolished by the IK Ca blocker NS6180 alone.Importantly, NO also contributed to endothelialhyperpolarization in RCA, as L-NAME and ADMA each reduced ACh hyperpolarization and vasorelaxation.This inhibitory component was prevented by L-arginine.In RMA, although EDH loss enhanced depolarization and vasoconstriction to the adrenergic agonist PE, it did not enable spike potentials, as NO was still available to suppress T-type VGCCs. 14In contrast with L-NAME, we show ADMA failed to block RMA NO-mediated vasorelaxation, although it did increase VSM electrical excitability.This suggests less NO is required for vasorelaxation than to suppress Ca 2+ -based depolarization.ADMA is metabolized by intracellular DDAH, which has 2 isoforms DDAH1 and DDAH2. 17,23DDAH2 cannot metabolize ADMA, but metabolism by DDAH1 has a significant influence on arterial function, as block with S-2-amino-4(3-methylguanidino)butanoic acid (412W) induced progressive vasoconstriction in rat aorta, which was reversed by exogenous L-arginine.412W also reversed the loss of endothelium-dependent (NO) vasorelaxation in human saphenous artery. 17,30Both effects demonstrate continual turnover of methylarginines, with DDAH1 ensuring the intracellular ADMA accumulation is not normally sufficient to block NO synthesis.We used the recently developed and selective DDAH1 inhibitor, L-257, which binds within the active site of the enzyme elevating ADMA sufficiently to inhibit NO signaling. 23n RMA, the ability of L-257 to inhibit ADMA-resistant ACh vasorelaxation suggests that ADMA DDAH1metabolism normally protects NO synthase activity.This profile was similar in wire-mounted and pressurized RMA, and 1 mmol/L L-arginine prevented the inhibitory action of ADMA±L-257.Although DDAH1 has specific affinity for ADMA (and N G -monomethyl L-arginine), it does not degrade L-NAME, consistent with the divergent effects obtained with each inhibitor in RMA. 31 The profile of inhibition in RCA was different.Once EDH was blocked, either ADMA or L-NAME totally abolished ACh vasorelaxation.Low levels of DDAH1 will predispose RCA to ADMA accumulation, block of NOS and enhanced vasoreactivity, it also indicates methylarginine metabolism varies between vascular beds.
Overall, the present experiments show the endogenous methylarginine, ADMA can enhance small artery vasoreactivity in a similar way to the synthetic NO synthase inhibitor L-NAME.In both cases, increased vasoreactivity is due to VSM electrical excitability developing on loss of NO.However, as only male rats were used in the present study, to limit inter-sex variability, these conclusions require verification in females.The importance of our observations is contextualized by a large literature supporting a fundamental role for ADMA in CVD, when plasma levels increase from low to mid micromolar concentrations and ADMA is considered an independent predictor of morbidity and mortality. 2,4While this range is close to NO synthase K i , plasma concentrations of L-arginine are greater (>100 µmol/L and mmol/L intracellularly) questioning the importance of ADMA in CVD.However, arginine supplementation enhances NO bioavailability, referred to as the "arginine paradox".Enhanced NO is attributed to arginine overcoming constitutive NOS inhibition by ADMA.Close to the site of synthesis, intracellular concentrations of ADMA will be far greater than in plasma, which may explain these observations. 233][34] Thus, ADMA accumulation may be a major contributor to the decline in NO causing microvascular dysfunction, including coronary microvascular dysfunction.In vasospastic angina, reduced amino acid transporter activity has been linked to ADMA accumulation, eNOS uncoupling, and systemic endothelial dysfunction. 350][41] Widespread dysfunction across the vasculature is consistent with declining endothelial NO bioavailability. 42

PERSPECTIVES
In large arteries, NO is the predominant endotheliumdependent vasodilator and the increased vasoreactivity that develops in CVD has been ascribed to loss of NO-vasodilator capacity.The VSM cells in small resistance arteries have a greater density of L-type VGCCs, and vasodilation is dominated by EDH-linked changes in membrane potential.As a result, EDH can sustain endothelium-dependent vasodilation in the absence of NO, although less so in coronary arteries where NO contributes to EDH.As well as vasodilation, we show NO directly suppresses VSM reactivity and reduce bioavailability with the naturally occurring NO synthase inhibitor ADMA enables spontaneous depolarizing spikes and vasospasm in small coronary arteries.Our data also suggest the capacity of DDAH1 to metabolize ADMA is less in RCA than other parts of the circulation, potentially predisposing these arteries to increased vasoreactivity/vasospasm as ADMA accumulates.It is, therefore, important to consider increased vasoreactivity as a change in the VSM, not simply as a loss of NO-vasodilator capacity.As NO chronically suppresses VSM excitability, opposing or reversing NO loss may offer an effective approach to address coronary microvascular dysfunction and possibly the development of CVD.We previously suggested selective T-type VGCC block as a strategy to counter enhanced VSM electrical activity, without abolishing the myogenic tone necessary for blood flow autoregulation as the latter is underpinned by L-type VGCCs. 14An alternative/additional possibility is to enhance/protect NO signaling with L-arginine supplementation or raise cyclic guanosine monophosphate with selective phosphodiesterase 5 inhibitors.Both would avoid long-term use of nitrates to generate NO and the associated complications from tolerance and undesirable side effects that include nitrosative stress. 43

Figure 5 .
Figure 5. Rapid depolarizing spikes and vasospasm during exposure to asymmetric dimethylarginine (ADMA) reversed by L-arginine in coronary arteries.A, Representative traces of vascular smooth muscle (VSM) spikes (top) and enhanced vasoconstriction (lower) with 300 µmol/L ADMA.Gray dotted lines, membrane potential and myogenic tone before ADMA.B, Box plot summarizing the amplitude of depolarizing spikes and vasoconstriction as mean±SEM for the maximum and minimum values of mV and mN/mm with sample mean.Membrane potential and tension before myogenic tone developed (n=22), with myogenic tone (MT, n=19), with 300 µmol/L ADMA (n=6), ADMA+1 mmol/L L-Arg (n=5), 100 µmol/L N G -monomethyl L-arginine methyl ester (L-NAME; n=12).**P<0.01vs MT.C, Fourier transform showing mean power of waveform during depolarization/spikes (upper) and associated vasoconstriction (lower); n, as above in B. D, Mean power from each waveform in C. *P<0.05 vs MT, **P<0.01vs MT, ***P<0.001vs MT, ### P<0.05 vs ADMA.All statistical tests were 1-way ANOVA with Bonferroni multiple comparisons, except for tension in D, where Kruskal-Wallis test with Dunn multiple comparisons was performed.AUC indicates area under curve; and Em, VSM membrane potential.Downloaded from http://ahajournals.org by on April 19, 2024